Tangential Flow Filtration Device Research Articles

Electric Field Assisted Tangential Flow Filtration Device for Highly Effective Isolation of Bioactive Small Extracellular Vesicles from Cell Culture Medium.

Small extracellular vesicles (sEVs) are proven to hold great promise for diverse therapeutic and diagnostic applications. However, batch preparation of sEVs with high purity and bioactivity is a prerequisite for their clinical translations. Herein, we present an electric field assisted tangential flow filtration system (E-TFF), which integrates size-based filtration with electrophoretic migration-based separation to synergistically achieve the isolation of high-quality sEVs from cell culture medium. Compared with the gold-standard ultracentrifugation (UC) method, E-TFF not only improved the purity of sEVs by 1.4 times but also increased the yield of sEVs by 15.8 times. Additionally, the entire isolation process of E-TFF was completed within 1 h, about one-fourth of the time taken by UC. Furthermore, the biological activity of sEVs isolated by E-TFF was verified by co-incubation of sEVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) with HT22 mouse hippocampal neuronal cells exposed to amyloid-β (Aβ). The results demonstrated that the sEVs isolated by E-TFF exhibited a significant neuroprotective effect. Overall, the E-TFF platform provides a promising and robust strategy for batch preparation of high-quality sEVs, opening up a broad range of opportunities for cell-free therapy and precision medicine.

Membrane-Based Hybrid Method for Purifying PEGylated Proteins

PEGylated proteins are usually purified using chromatographic methods, which are limited in terms of both speed and scalability. In this paper, we describe a microfiltration membrane-based hybrid method for purifying PEGylated proteins. Polyethylene glycol (or PEG) is a lower critical solution temperature polymer which undergoes phase transition in the presence of a lyotropic salt and forms micelle-like structures which are several microns in size. In the proposed hybrid method, the PEGylated proteins are first converted to their micellar form by the addition of a lyotropic salt (1.65 M ammonium sulfate). While the micelles are retained using a microfiltration membrane, soluble impurities such as the unmodified protein are washed out through the membrane. The PEGylated proteins thus retained by the membrane are recovered by solubilizing them by removing the lyotropic salt. Further, by precisely controlling the salt removal, the different PEGylated forms of the protein, i.e., mono-PEGylated and di-PEGylated forms, are fractionated from each other. Hybrid separation using two different types of microfiltration membrane devices, i.e., a stirred cell and a tangential flow filtration device, are examined in this paper. The membrane-based hybrid method for purifying PEGylated proteins is both fast and scalable.

Shear ignorance? Think again: breaking the perception of shear within viral vector manufacturing

Charlotte Barker,  Editor, BioInsights, speaks to MilliporeSigma’s Ratish Krishnan, Senior Strategy Consultant, Bioprocessing Strategy Operationalization (pictured left) and Akshat Gupta, Associate Director, Global Biopharma Center of Excellence (pictured right).The tangential flow filtration (TFF) unit operation in viral vector manufacturing is a critical step on the path to commercialization. In this episode, Ratish Krishnan and Akshat Gupta discuss best practices – and common misconceptions – when establishing process conditions and utilizing different TFF device formats.

Alpha-2-macroglobulin as a novel diagnostic biomarker for human bladder cancer in urinary extracellular vesicles.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p<0.0001). There was no significant difference in whole urine a2M levels between patients with BC and controls (p=0.317). We observed that compared to classical differential centrifugation, ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method for uEV protein analysis. We expect that our approach for EV analysis will provide an efficient route for the identification of clinically meaningful uEV-based biomarkers for cancer diagnosis.

MP06-17 ALPHA-2-MACROGLOBULIN IN URINARY EXTRACELLULAR VESICLES IS A NOVEL PROTEIN MARKER FOR DIAGNOSIS OF HUMAN BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-17 ALPHA-2-MACROGLOBULIN IN URINARY EXTRACELLULAR VESICLES IS A NOVEL PROTEIN MARKER FOR DIAGNOSIS OF HUMAN BLADDER CANCER Jinsung Park, Jisu Lee, Yun Hee Kang, Ji Young Mun, Hyun-Woo Oh, Yoon-Kyoung Cho, and Myung-Shin Lee Jinsung ParkJinsung Park More articles by this author , Jisu LeeJisu Lee More articles by this author , Yun Hee KangYun Hee Kang More articles by this author , Ji Young MunJi Young Mun More articles by this author , Hyun-Woo OhHyun-Woo Oh More articles by this author , Yoon-Kyoung ChoYoon-Kyoung Cho More articles by this author , and Myung-Shin LeeMyung-Shin Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary extracellular vesicles (uEVs) are considered potential biomarkers for bladder cancer (BC). However, challenges associated with the effective isolation and analyses of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we sought to identify novel uEV-derived diagnostic biomarkers for BC. METHODS: Urine samples were obtained between July 2018 and October 2020. 1) To identify optimal uEV isolation methods, three different methods (differential centrifugation, ExoLutE®, ExoDisc) were compared by various methods. 2) Through comparative analysis of uEVs obtained from BC patients pre- and post-operation using an antibody array, candidate uEV biomarkers were selected. 3) Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from 83 patients (23 control subjects and 60 BC patients) validated the clinical significance of the uEV biomarkers. RESULTS: We found that ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method than other methods (Fig 1). In the analysis of pre-and post-operative uEVs from BC patients by the ExoDisc, three proteins, namely cadherin-13, clusterin, and alpha-2-macroglobulin (A2M), were identified as potential uEV biomarkers, while A2M exhibited the most significant difference. In the validation study, the A2M expression in uEVs from BC patients was significantly higher than the control group (Fig 2A) with good diagnostic performance (Fig 2B). The uEV A2M expression in the high-grade BC was significantly higher than that in low-grade BC (Fig 2C), whereas no significant difference were detected in the whole urine samples (Fig 2D). The uEV A2M expression with a cut-off of 0.035 (highest Youden index) robustly discriminated BC patients from controls, with a sensitivity of 93.3%, a specificity of 34.8%. CONCLUSIONS: We demonstrated the potential of A2M as a novel uEV biomarker for BC diagnosis through an optimized EV isolation and analysis workflow. Source of Funding: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01060913) to Dr. Jinsung Park © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e83 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jinsung Park More articles by this author Jisu Lee More articles by this author Yun Hee Kang More articles by this author Ji Young Mun More articles by this author Hyun-Woo Oh More articles by this author Yoon-Kyoung Cho More articles by this author Myung-Shin Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Critical flux behavior of ultrathin membranes in protein-rich solutions

Critical flux defines the ideal operating conditions in tangential flow filtration (TFF). Below this value a TFF process can, in principle, proceed indefinitely without membrane fouling. The critical flux behavior of ultrathin membranes, which are particularly valuable for small volume processing, has not yet been explored. Here we investigate the critical flux behavior of ultrathin nanoporous silicon nitride (NPN) membranes in concentrated protein solutions using a microfluidic TFF device. We find that NPN (~19% porosity; 60 nm average pore size; 100 nm thick) has the ability to process bovine serum albumin (BSA) solutions as concentrated as 10 mg/mL at 30 μL/min without exceeding a critical flux threshold. At 30 mg/mL and 60 mg/mL, NPN enables 40% and 20% of the supply to be filtered without fouling. In these experiments, NPN membranes achieve critical flux values as high as ~1200 LMH, which is 5x larger than 0.2 μm polyethersulfone (PES) membranes processing 5 mg/mL BSA and an order of magnitude higher than track-etch polymeric membranes (10 μm thick; 80 nm pores) in direct comparisons. The effects of nanoparticle capture on critical flux are also studied here, simulating the capture of small extracellular vesicles (sEVs) in forthcoming diagnostic applications. Our work not only demonstrates the utility of ultrathin membranes for processing concentrated protein solutions, it illustrates the value of applying membrane science principles developed for macroscale systems (e.g. critical flux, TFF) to the micro/nano scale.

An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes.

The availability of material for experimental studies is a key constraint in the development of full‐scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale‐down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale‐down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full‐scale studies. In this study the performance of a pilot‐scale tangential flow filtration (TFF) system based on a membrane flat‐sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm2 of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot‐scale TFF device operating at ~500‐fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate‐pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.

Collection of in vivo-like liver cell secretome with alternative sample enrichment method using a hollow fiber bioreactor culture system combined with tangential flow filtration for secretomics analysis

A hollow fiber bioreactor (HFB) culture system coupled with a tangential flow filtration (TFF) device was used for HepG2 cell secretome analysis. In order to reduce the loss of low-molecular-weight proteins, two new features, the hollow fiber with 0.1 μm pore size and a TFF device with a membrane of 1 kDa molecular weight cutoff, were added to the system described previously. The HFB culture system and the conventional dish culture method for secretome collection were compared side by side. It was observed that only a small fraction of cells (<0.01%) were lysed in the HFB culture system, in contrast to the 2.73% in the conventional dish culture. A total of 111 proteins were identified in the collected conditioned medium (CM) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with this improved collection procedure. Many of these proteins reported to be biomarkers for liver-related diseases. About 16% of the identified proteins were smaller than 20 kDa, demonstrating that the modified collection system had the ability to reduce the loss of low-molecular-weight proteins, in contrast to our previous collection system. The percentage increase of proteins classified as extracellular space or plasma membrane between the conventional dish culture and the HFB culture system was 40–60%. We believed that in vivo-like culture environments could support liver cells to improve protein secretion than conventional dish cultures. We suggest that the combination of the HFB culture system, TFF device, and LC–MS/MS analysis, would be an efficient procedure for the collection and characterization of in vivo-like cell secretome.

High Performance Tangential Flow Filtration Using Charged Affinity Ligands

High performance tangential flow filtration is an emerging technology for protein separations. The objective of this work was to examine the use of small charged affinity ligands to control the protein charge and thus extend the potential applicability of this separation technique. Experiments were performed using binary mixtures of bovine serum albumin and ovalbumin, with Cibacron Blue added as an affinity ligand. The addition of Cibacron Blue caused a large increase in the selectivity due to the increase in BSA retention associated with the strong electrostatic repulsion between the negatively‐charged protein‐ligand complex and the negatively‐charged cellulosic membrane. Appropriate conditions for the separation were identified using total recycle experiments in a small‐scale tangential flow filtration device. A diafiltration process was designed to separate BSA from ovalbumin, with the Cibacron Blue recovered in a second stage operated with a buffer containing NaSCN to displace the dye from the protein. The results demonstrate the ability to use small charged affinity ligands with bio‐specific binding characteristics to enhance separations by high performance tangential flow filtration.

Two Anti-radial Spoke Monoclonal Antibodies Inhibit Chlamydomonas Axonemal Motility by Different Mechanisms

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

High-Titer Adenovirus Vector Production in 293S Cell Perfusion Culture

Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production.

On-line monitoring and control of fermentation processes by flow-injection analysis

On line flow-injection analysis (FIA) systems were developed for the real-time measurement and control of concentrations of small molecules (i.e. MW < 500) in fermentation media. An on-line tangential flow filtration device was developed to provide a particle-free sample stream to the analyzers. Analysis of fermentation supernatant is problematic because it is a complex and variable sample matrix. Using FIA techniques, in-line sample pretreatment strategies have been implemented to eliminate specific interferences. Two examples, demonstrating the use of on-line FIA for monitoring acetate and for controlling phosphate in E. coli fermentations, are discussed.

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.

A filtration method for harvesting Mycoplasma pneumoniae cells

An easy method for harvesting Mycoplasma pneumoniae ( M.pn.) cells from batch cultures by a tangential flow filtration device is described. The quality of the resulting cell suspension is monitored by two common serological tests; complement fixation test (CFT) and enzyme-linked immunosorbent assay (EIA). For CFT a lipid extract of the M.pn cell suspension is used as antigen and the remaining lipid-depleted cells are the antigen for EIA. No difference is observed between this lipid antigen and the commercial one when the respective CFT results are compared. The ‘protein antigen’ seems to be sensitive for detecting titer rises between paired sera of patients with an enfectious illness. An additional advantage is that the EIA allows detection of antibodies belonging to different immunoglobulin classes.

Production of biosurfactants by a mixed bacteria population grown in continuous culture on crude oil

Large amounts (3 g.l−1) of tensio-active substances can be produced continuously and recuperated using a tangential-flow filtration device. These compounds can emulsify crude oil and the emulsions, obtained with a crude oil over biosurfactants ratio of 500, remain stable for hours.